Urokinase-type plasminogen activator receptor (uPAR) is over-expressed in a variety of human cancers1, including prostate cancer (PC), where uPAR expression in tumor biopsies and shed forms of uPAR in plasma have been found to be associated with advanced disease and poor prognosis2-9. Moreover, in patients with localized PC, high preoperative plasma uPAR levels have been shown to correlate with early progression10. Consistent with uPARs important role in cancer pathogenesis, through extracellular matrix degradation facilitating tumor invasion and metastasis, uPAR is considered an attractive target for both therapy11-13 and imaging 14 and the ability to non-invasively quantify uPAR density in vivo is therefore crucial.
Radiolabeling and in vivo evaluation of a small peptide radiolabeled with Cu-6415 and Ga-6816 have been described for PET imaging of uPAR in various human xenograft cancer models. Such tracers could specifically differentiate between tumors with high and low uPAR expression and furthermore established a clear correlation between tumor uptake of the uPAR PET probe and the expression of uPAR15. However, 18F (t1/2=109.7 min; β+, 99%) is considered the ideal short-lived PET isotope for labeling of small molecules and peptides due to the high positron abundance, optimal half-life and short positron range.
Recently, an elegant one step radiolabeling approach was developed for radiofluorination of both small peptides and proteins based on complex binding of (Al18F)2+ using 1,4,7-triazacyclononane (NOTA) chelator17-20. In this method, the traditional critical azeotropic drying step for 18F-fluoride is not necessary, and the labeling can be performed in water. A number of recently published studies have illustrated the potential of this new 18F-labeling method, where successful labeling of ligands for PET imaging of angiogenesis21,22, Bombesin23, EGFR24 and hypoxia25 have been demonstrated.
Various radio-labelled peptide compositions have been developed or are under development for site-specific targeting of various antigens, receptors and transporters for PET imaging. The general principle involves attaching a selected positron emitting radionuclide to a peptide and/or protein having a high specificity for a particular antigen for visualize and quantify the expressing and/or activity level using PET imaging. This field of research has shown particular applicability for tumor diagnosis, staging and treatment monitoring. A particularly desirable tumor antigen is uPAR in many different solid tumors including but not limited to non-small cell lung carcinomas, brain tumors, prostate tumors, breast tumors, colorectal tumors, pancreatic tumors and ovarian tumors.
DOTA (1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10 tetraazacyclo dodecane) and its derivatives constitute an important class of chelators for biomedical applications as they accommodate very stably a variety of di- and trivalent metal ions. An emerging area is the use of chelator conjugated bioactive peptides for labelling with radiometals in different fields of diagnostic and therapeutic nuclear oncology. NOTA and its derivatives constitute another important class of chelators for biomedical applications.
uPAR PET imaging has been exploited in several human cancer xenograft models using a small linear DOTA-conjugated peptide, DOTA-AE105 radiolabeled with 64Cu (Li et al, 2008, Persson et al, 2011) and 68Ga (Persson et al, 2012) and using NODAGA (NODAGA-AE105) radiolabeled with 68Ga (Persson et al, 2012).
Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules for cell surface-associated plasminogen activation both in vitro and in vivo. High expression of uPA and uPAR in many types of human cancers correlate with malignant tumor growth and associate with a poor prognosis, possibly indicating a causal role for the uPA/uPAR system in cancer progression and metastasis. Studies by immunohistochemistry and in situ hybridization indicate that expression levels of the components from the uPA system are generally very low in normal tissues and benign lesions. It has also been reported that the uPA/uPAR system is involved in regulating cell-extracellular matrix interactions by acting as an adhesion receptor for vitronectin and by modulating integrin function. Based on these properties, the uPA/uPAR system is consequently considered an attractive target for cancer therapy.
WO 01/25410 describes diagnostically or therapeutically labelled uPAR-targeting proteins and peptides. The peptide or protein comprises at least 38 amino acid residues, including residues 13-30 of the uPAR binding site of uPA.
U.S. Pat. No. 6,277,818 describes uPAR-targeting cyclic peptide compounds that may be conjugated with a diagnostic label. The peptides are based on the amino acid residues 20-30 of uPA.
U.S. Pat. No. 6,514,710 is also directed to cyclic peptides having affinity for uPAR. The peptides may carry a detectable label. The peptide comprises 11 amino acids joined by a linking unit.
Ploug et al. in Biochemistry 2001, 40, 12457-12168 describes uPAR targeting peptides but not in the context of imaging, including amino acid sequences as described in the present document. Similar disclosure is provided in U.S. Pat. No. 7,026,282.
The efficient targeting of uPAR demands a selective high-affinity vector that is chemically robust and stable.